The invention finds application in the field of structural identification of oxidation products, for example by mass spectrometry, NMR, infrared. Notably, in the pharmaceutical field, the invention applies to the making of pharmacological and toxicological tests, more specifically for studying the oxidative fate of certain drugs with view to evaluating the stability, the chemical activity and the biological reactivity of the main intermediate species. Also, in the field of the agri-food industry, the invention applies to elucidation of oxidative or photo-oxidative degradation of food additives such as preservatives, coloring agents, antioxidants. In further another field, that of the environment, the invention applies to predicting the fate of emerging pollutants like drugs, detergents, phenol derivatives.
Prediction of risks posed by many chemical entities (such as xenobiotics) for human health and its environment is today considered as a major social issue. Many xenobiotics like drugs, emergent pollutants, pesticides, preservatives, food additives and other substances, have shown that they may cause major secondary effects, as testified by examples of withdrawal of certain drugs, food preservatives and plant protection products on the market.
In this context, the development of novel analytic tools in vitro mimicking oxidative metabolism is presently an emerging axis of vital investigation for predicting possible toxic effects of chemical species. These novel tools are essentially based on the prediction of oxidative degradation schemes which a xenobiotic may undergo (Donato, M. T.; Castell, J. V.; Gomez-Lechon, M. J., Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models. Journal of Hepatology 1999, 31, (3), 542-549; Dong H., Haining, R. L., Thummel, K. E.; Rettie, A. E.; Nelson, S. D.; Involvement of human cytochrome p450 2D6 in the bioactivation of acetaminophen. Drug Metab Dispos 2000, 28, (12), 1397-400; Ferchaud, V; Le, B. B., Montrade, M-P.; Maume, D.; Monteau, F.; André, F., Gas chromatographic-mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration. J. Chromatogr., B Biomed. Sci Appl. 1997, 695, (2) 269-277).
Several biological models used in vitro have been explored for studying oxidative metabolization of xenobiotics (Henderson, M. C, Siddens, L. K, Morré, J. T, Krueger, S. K, Williams, D. E. Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes. Toxicology and Applied Pharmacology 2008, 233, (3), 420-427; Yun, C-H. Miller, G. P, Guengerich, F. P. Rate-Determining Steps in Phenacetin Oxidations by Human Cytochrome P450 1A2 and Selected Mutants. Biochemistry 200, 39, (37), 11319-11329).
For example mention may be made of the use of liver sections for studying certain metabolisms with view to identifying different metabolites. The use of hepatocytes, today commercially available, has also been highly successful in this field.
Further, by the development of molecular biology and the launching on the market of many recombinant enzymes, a more significant preference is today described for using enzymes from the family of P450 cytochromes (Dong H., Haining, R. L., Thummel, K. E.; Rettie, A. E.; Nelson, S. D.; involvement of human cytochrome p450 2D6 in the bioactivation of acetaminophen. Drug Metab Dispos 2000, 28, (12), 1397-400; Anzenbacher, P. Anzenbacherova, E. Cytochromes P450 and metabolism of xenobiotics. Celle. Mol. Life Sci. 2001, 58, (5/6), 737-747; Delaforge, M. Pruvost, A. Perrin, L. Andre, F. Cytochrome P450-mediated oxidation of glucuronide derivatives: example of estradiol-17Î2-glucuronide oxidation to 2-hydroxy-estrdiol-17Î2-glucuronide by CYP 2C8 Drug Metab Dispos 2005, 33, (3), 466-473; Isin, E. M. Guengerich, F. P., Complex reactions catalyzed by cytochrome P450 enzymes Biochimica and Biophysica Acta (BBA)—General Subjects 2007, 1770, (3), 314-329).
These biological models are considered as tools of choice for studying oxidative metabolism; they not only give the possibility of providing a new understanding of the oxidative routes, but also of elucidating the mode of action or explaining the reasons of a possible toxicity of a chemical entity. These tests in vitro, of highly widespread use in the pharmaceutical industry and in many research laboratories, are simplified models as compared with tests in vivo, and give the possibility of setting up bases of experimental models in vivo, notably in the case of development of candidate drugs, and in the case of studying the effects of emerging pollutants on human health and on the environment.
It should also be noted that the development of tests in vitro in the field of oxidative degradation during the last decade also owes its success to the development of analytic instrumentation within these tests themselves, with the use of techniques such as extraction techniques (SPE), increasingly performing columns, HPLC coupling and mass spectrometry.
However, their advantage does not annihilate certain constraints inherent to the techniques used in vitro: slow analysis, difficulty of structurally characterizing the intermediate species stemming from the oxidative degradation of a xenobiotic (small generated amounts), low compatibility of organic solvents (solubilization of xenobiotics) with the use of biological materials (cells, enzymes and other materials) . . . .
Some of these problems were circumvented by using chemical methods (Chorghade, M. S.; Hill, D. R.; Lee, E. C.; Pariza, R. J.; Dolphin, D. H.; Hino, F.; Zhang, L.-Y., Metalloporphyrins as chemical mimics of cytochrome P-450 systems. Pure Appl. Chem. 1996, 68, (3), 753-756) and electrochemical methods (Karst, U.; Diehl, G.; Hayen, H. Coupling electrochemistry to mass spectrometry and high performance liquid chromatography. 2003; Karst, U., Analytical methods: Electrochemistry/mass spectrometry (EC/MS)—a new tool to study drug metabolism and reaction mechanisms. Angew. Chem., Int. Ed. 2004, 43, (19), 2476-2478).
Indeed, it has been shown that a conventional electrochemical cell (EC) with three electrodes associated with the performances of liquid phase chromatography (LC) and of mass spectrometry (EC-LC-MS coupling) may mimic certain reactions of oxidative metabolism, notably those initiated and catalyzed by the family of P450 cytochromes, such as for example N-dealkylation, O-dealkylation, epoxidization, oxidation of thiols, of alcohols, dehydrogenation of aromatic rings (Nouri-Nigjeh, E. Permentier, H. P. Bischoff, R. Bruins, A. P., Electrochemical Oxidation by Square-Wave Potential Pulses in the Imitation of Oxidative Drug Metabolism. Anal. Chem. 2011, (83), 14, 5519. Nouri-Nigjeh, E. Bruins, A. P. Bischoff, R. Permentier, H. P., Electrocatalytic oxidation of hydrogen peroxide on a platinum electrode in the imitation of oxidative drug metabolism of lidocaine. Analyst. 2012, (137), 4698.).
However, many points still remain to be explored. For example, we notice a lack of a device for synthesizing in a sufficient amount and under a stable condition main intermediate species from oxidative degradation of a xenobiotic. This limits the use of NMR on the one hand for elucidating with more accuracy the chemical structure of the different species, and the application of tests for predetermining the threshold concentration for evaluating the inhibition or toxic potential of the main species from oxidation of a xenobiotic on the other hand.
One of the objects of the invention is therefore to provide a solution to the aforementioned problems and drawbacks.
The invention thus according to a first aspect relates to a device for synthesizing intermediate species of a chemical entity, electrochemically.